Our brain is made up of intricate networks of over 85 billion interconnected neurons, making it one of the most complex objects in the universe. The thousands of connections made by each of these neurons lay the foundation of how we perceive, interact with our environment and lead our existence. Neural circuits are wired up during development by virtue of a molecular code that allows individual neurons to find their correct connection partners. Given their functional significance, even subtle defects in the developmental mechanisms establishing synaptic connections are thought to underlie many neurodevelopmental disorders including autism spectrum disorders, Tourette syndrome, schizophrenia and epilepsy. Therefore, investigating the molecular mechanisms underlying the assembly of the connectome of functional neural circuits is highly significant. This proposal is the first to identify a new function for the axon guidance proteins Robo/Slit as synaptogenic molecules underlying the formation of excitatory synapses in hippocampal circuits important for learning and memory. Using an in vitro hemisynapse assay, we found that Robo1/2 are able to induce excitatory synapse formation in a Slit-dependent manner. We provide in vitro and in vivo evidence that Robo receptors are required postsynaptically, but not in presynaptic axons, to induce synaptogenesis. We also obtained preliminary results strongly suggesting that presynaptic Neurexins are functional receptors mediating Robo/Slit-dependent synaptogenesis. Developmental, conditional knockout of Robo2 from hippocampal CA1 PNs leads to a drastic reduction of excitatory synapse number in the adult. We propose to test the biochemical nature of this novel, potentially tripartite Robo/Slit/Neurexin transsynaptic adhesion complex and use this molecular effectors of synaptic specificity to test the functional consequences of its disruption on CA1PNs spatial tuning properties. Specifically, we will employ chronic in vivo 2P Ca2+ imaging at cellular resolution to monitor functional properties of spatial information encoding following developmental Robo2 conditional deletion. We will complement this approach with rigorous behavioral assessment of Robo2 CA1-specific conditional deletion with a focus on hippocampus-dependent spatial learning behaviors. Altogether, in an unprecedented multidisciplinary approach, we will directly test the role of Slit-Robo-dependent synaptogenesis on the establishment of functional circuits in vivo.